Efficient method for nuclear reprogramming

ABSTRACT

A method of preparing induced pluripotent stem cells, comprising a nuclear reprogramming step with a nuclear reprogramming factor in the presence of miRNA, wherein said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence thereof.

PRIOR RELATED APPLICATIONS

This application is a continuation-in-part of PCT/JP2008/59586, filedMay 23, 2008, which claims priority of U.S. Provisional Application No.60/996,893, filed Dec. 10, 2007, and this application also claimspriority of U.S. Provisional Application No. 60/996,893, filed Dec. 10,2007. The entire disclosures of each of the above-cited applications areconsidered as being part of this application and are expresslyincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to efficient methods for preparing inducedpluripotent stem cells through reprogramming of somatic cells, toinduced pluripotent stem cells, to uses of induced pluripotent stemcells and to somatic cells derived by inducing differentiation of saidpluripotent cells. The present invention also relates to nuclearreprogramming factors and to miRNAs involved in generating inducedpluripotent stem cells. The present invention also relates to screeningmethods, treatments, and therapies involving the use of inducedpluripotent stem cells.

BACKGROUND OF THE INVENTION

Embryonic stem cells (ES cells) are stem cells established from human ormouse early embryos which have a characteristic feature that they can becultured over a long period of time while maintaining pluripotentability to differentiate into all kinds of cells existing in livingbodies. Human embryonic stem cells are expected for use as resources forcell transplantation therapies for various diseases such as Parkinson'sdisease, juvenile diabetes, and leukemia, taking advantage of theaforementioned properties. However, transplantation of ES cells has aproblem of causing rejection in the same manner as organtransplantation. Moreover, from an ethical viewpoint, there are manydissenting opinions against the use of ES cells which are established bydestroying human embryos.

If dedifferentiation of patients' own differentiated somatic cells couldbe induced to establish cells having pluripotency and growth abilitysimilar to those of ES cells (these cells are referred herein to as“induced pluripotent stem cells” or “iPS cells,” though they aresometimes called “embryonic stem cell-like cells” or “ES-like cells”),it is expected that such cells would be useful as ideal pluripotentcells, free from rejection or ethical difficulties. Recently, it hasbeen reported that such iPS cells can be produced from differentiatedcells of mouse or human, which has created a great sensation(International Publication No. WO2007/069666 A1; Takahashi et al., Cell126:663-76, 2006; Takahashi et al., Cell 131:861-72, 2007; Yu et al.,Science 318:1917-20, 2007; and Park et al., Nature 451:141-46, 2008,herein incorporated by reference in their entireties). Thus, the term“induced pluripotent stem cells (iPS cells)” refers to cells havingsimilar properties to those of ES cells, and more specifically the termincludes undifferentiated cells which are reprogrammed from somaticcells and have pluripotency and proliferation potency. However, thisterm is not to be construed as limiting in any sense, and should beconstrued to have its broadest meaning.

These methods include a reprogramming step through introduction of aplurality of specific factors (for example, four factors of Oct3/4,Sox2, Klf4, and c-Myc can be used in Takahashi et al., Cell 126:663-76,2006), and the introduction of these factors is mediated by viralvectors such as retroviral or lentiviral vectors. However, allpreviously reported nuclear reprogramming methods mediated by theintroduction of genes involve a problem of low efficiency in which onlya small number of induced pluripotent stem cells can be obtained. Inparticular, there is a problem in that, if reprogramming is carried outin somatic cells through the introduction of three factors (namely,Oct3/4, Sox2, and Klf4) excluding c-Myc, then the production efficiencyof induced pluripotent stem cells becomes low. Nevertheless, theefficient production of iPS cells without the use of c-Myc would providecertain advantages, as c-Myc is suspected to cause tumorigenesis intissues and in chimeric mice generated from induced pluripotent stemcells.

It is known that various small RNAs are expressed in cells. Examples ofsmall RNA include RNA molecules of about 18-25 nucleotides in lengthwhich can be cleaved out with a dicer, an RNase specific todouble-stranded RNA. Small RNA is mainly classified into siRNA (smallinterfering RNA) and miRNA (microRNA, hereinafter abbreviated as“miRNA”). Small RNA is known to function as a guide molecule for findingtarget sequences in processes such as translational suppression, mRNAdegradation, or alteration of chromatin structure. Small RNAs functionvia RNA interference (RNAi) or miRNA molecular mechanisms. In addition,small RNA is also known to play an important role in the regulation ofdevelopmental processes (for example, as general remarks, refer toJikken Igaku (Experimental Medicine), 24, pp. 814-819, 2006; andmicroRNA Jikken Purotokoru (microRNA Experimental Protocol), pp. 20-35,2008, YODOSHA CO., LTD., herein incorporated by reference in theirentireties).

ES cell-specific microRNAs have been identified (Houbaviy et al.,Developmental Cell 5:351-58, 2003). In particular, ES cell-specificexpression of a microRNA cluster, which includes several types of miRNAsin mouse ES cells, has been reported (Houbaviy et al., DevelopmentalCell 5:351-58, 2003, herein incorporated by reference in its entirety).It has also been reported that miRNA-295 suppressed the expression ofRb12, a member of the Rb tumor suppressor gene family, and increased theexpression of methylase to be thereby associated with DNA methylation(Sinkkonen et al., Nature Structural & Molecular Biology 15:259-267,2008; Benetti et al., Nature Structural & Molecular Biology 15:268-279,2008, herein incorporated by reference in their entireties). However,these documents do not disclose any role of small RNA in the nuclearreprogramming of somatic cells.

SUMMARY OF THE INVENTION

The present invention relates to methods for efficiently preparinginduced pluripotent stem cells. The present invention provides methodsfor achieving efficient preparation of induced pluripotent stem cells inthe presence of miRNA. The present invention also provides methods forefficient preparation of induced pluripotent stem cells with a nuclearreprogramming factor. The present invention also provides methods forefficient preparation of induced pluripotent stem cells with a nuclearreprogramming factor in the presence of increased miRNA as compared tothe level present in the somatic cell prior to nuclear reprogramming.The present invention also provides such methods wherein the nuclearreprogramming factor does not include c-Myc and/or Sox2.

The invention provides a method of preparing induced pluripotent stemcells, comprising nuclear reprogramming at least one somatic cell withnuclear reprogramming factor and at least one miRNA, wherein the atleast one miRNA increases efficiency of the nuclear reprogramming of theat least one somatic cell compared to nuclear reprogramming of the atleast one somatic cell with the nuclear reprogramming factor in theabsence of the at least one miRNA.

The invention also provides such a method, wherein the at least onemiRNA is expressed in embryonic stem cells at a higher level than insomatic cells.

The invention also provides such a method, wherein a gene encoding thenuclear reprogramming factor and/or the at least one miRNA is introducedinto the at least one somatic cell.

The invention also provides such a method, wherein a vector comprisingthe gene and/or a vector encoding the at least one miRNA is introducedinto the at least one somatic cell.

The invention also provides such a method, wherein the vector comprisingthe gene or encoding the at least one miRNA is a retroviral vector.

The invention also provides such a method, wherein the gene is selectedfrom an Oct family gene, a Klf family gene, and a Sox family gene.

The invention also provides such a method, wherein the gene is selectedfrom Oct3/4, Klf4, and Sox2.

The invention also provides such a method, wherein the nuclearreprogramming factor comprises Oct3/4, Klf4, and Sox2.

The invention also provides such a method, wherein the at least onemiRNA is introduced into the at least one somatic cell as primary miRNA.

The invention also provides such a method, wherein the at least onemiRNA is introduced into the at least one somatic cell as pre-miRNA.

The invention also provides such a method, wherein the at least onemiRNA comprises at least one miRNA represented by SEQ ID NOs: 1 to 14.

The invention also provides such a method, wherein the at least onemiRNA comprises at least one miRNA contained in miRNA clusterhsa-miR-302-367 cluster.

The invention also provides such a method, wherein the at least onemiRNA regulates DNA methylation.

The invention also provides such a method, wherein the at least onemiRNA regulates de novo DNA methylation.

The invention also provides such a method, wherein the at least onemiRNA down-regulates DNA methylation.

The invention also provides such a method, wherein the at least onemiRNA comprises at least 10 contiguous nucleotides of at least one miRNArepresented by SEQ ID NOs: 1 to 14.

The invention also provides such a method, wherein the at least onemiRNA comprises at least 30 contiguous nucleotides of at least one miRNArepresented by SEQ ID NOs: 1 to 14.

The invention also provides such a method, wherein the at least onemiRNA comprises at least 60 contiguous nucleotides of at least one miRNArepresented by SEQ ID NOs: 1 to 14.

The invention also provides such a method, wherein the nuclearreprogramming factor does not include c-Myc and/or Sox2.

The invention also provides such a method, wherein the at least onemiRNA comprises hsa-miR-302-367 cluster miRNA.

The invention also provides such a method, wherein the nuclearreprogramming factor comprises an Oct family gene member, a Sox familygene member, and a Klf family gene member.

The invention also provides such a method, wherein the at least onemiRNA comprises mmu-miR-295/295* and mmu-miR-294/294*.

The invention also provides such a method, wherein the at least onemiRNA comprises hsa-miR-302-367 cluster, hsa-miR-371-373 cluster andhsa-miR-520c miRNA.

The invention also provides such a method, wherein the nuclearreprogramming factor comprises a Klf family gene, and an Oct familygene.

The invention also provides such a method, wherein the nuclearreprogramming factor further comprises a Myc family gene.

The invention also provides such a method, wherein the nuclearreprogramming factor further comprises a Sox family gene.

The invention also provides such a method, wherein the nuclearreprogramming factor further comprises a Sox family gene.

The invention also provides such a method, wherein the nuclearreprogramming factor comprises KLF4 and OCT3/4.

The invention also provides such a method, wherein the nuclearreprogramming factor excludes a Sox family gene.

The invention also provides such a method, wherein the nuclearreprogramming factor excludes a Myc family gene.

The invention also provides such a method, where the at least onesomatic cell comprises a plurality of somatic cells.

The invention also provides a method of increasing the efficiency ofnuclear reprogramming comprising: adding a nuclear reprogramming factorand at least one miRNA to at least one somatic cell so that the numberof induced pluripotent stem cells produced is greater than in theabsence of the added miRNA.

The invention also provides an induced pluripotent stem cell induced byreprogramming a somatic cell, wherein the reprogramming is performed byadding at least one miRNA and in the absence of eggs, embryos, orembryonic stem (ES) cells.

The invention also provides such an induced pluripotent stem cell,wherein the induced pluripotent stem cell is a human cell.

The invention also provides an induced pluripotent stem cell obtained bya method of preparing induced pluripotent stem cells, comprising nuclearreprogramming at least one somatic cell with nuclear reprogrammingfactor and at least one miRNA, wherein the at least one miRNA increasesefficiency of the nuclear reprogramming of the at least one somatic cellcompared to nuclear reprogramming of the at least one somatic cell withthe nuclear reprogramming factor in the absence of the at least onemiRNA.

The invention also provides an pluripotent stem cell obtained by amethod of increasing the efficiency of nuclear reprogramming comprising:adding a nuclear reprogramming factor and at least one miRNA to at leastone somatic cell so that the number of induced pluripotent stem cellsproduced is greater than in the absence of the added miRNA.

The invention also provides somatic cell derived by inducingdifferentiation of any of the above pluripotent stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of confirmation on the production efficiency ofinduced pluripotent stem cells through induction of nuclearreprogramming in mouse embryonic fibroblasts with a combination of threegenes comprising Oct3/4, Klf4, and Sox2 (this combination—represented as“3f”, “c-Myc(−)”, or OSK”—means that c-Myc was omitted from acombination of four genes comprising Oct3/4, Klf4, Sox2 and c-Myc, whichis highly efficient for nuclear reprogramming), in the presence ofvarious miRNAs. 3f+DsRed represents a combination where DsRed (Discosomasp. red fluorescent protein) as a control was added to the combinationof the aforementioned three genes. The results of three independentexperiments are shown. The graph shows the number of ES-like colonies inthe cells transduced with OSK with or without DsRed, or with variousmiRNAs.

FIG. 2 shows the production efficiency of induced pluripotent stemcells. The top row of images shows the results of nuclear reprogrammingin mouse tail tip fibroblasts (TTFs) when DsRed was added, as a control,to the combination of three genes comprising Oct3/4, Klf4, and Sox2 (acombination of three genes in which c-Myc was omitted from thecombination of four genes). The bottom row of images shows the resultsof induction of nuclear reprogramming in mouse TTFs with the combinationof three genes comprising Oct3/4, Klf4, and Sox2 in the presence ofmmu-miR-295. The number in the figure indicates the number of Nanog GFPpositive colonies/the number of total colonies on days 7, 21, and 28after drug selection was started.

FIG. 3 shows the results of confirmation on the production efficiency ofinduced pluripotent stem cells through induction of nuclearreprogramming in adult human dermal fibroblasts expressing the mouseecotropic virus receptor Slc7a1 gene using lentivirus (aHDF-Slc-7a1)with the combination of three genes comprising Oct3/4, Klf4, and Sox2(Myc(−)3f: a combination of three genes in which c-Myc was omitted fromthe combination of four genes comprising Oct3/4, Klf4, Sox2, and c-Myc),or the combination of four genes comprising Oct3/4, Klf4, Sox2, andc-Myc (Y4f), in the presence of various miRNAs.

FIGS. 4A-B shows the results of ES-like colonies produced aftertransduction with 4 factors, i.e., OCT3/4, SOX2, KLF4, and c-MYC (OSMK),as well as with 3 factors, i.e., OCT3/4, SOX2, KLF4 in the presence ofvarious miRNAs (OSK+). FIG. 4A shows the number of human ES-likecolonies obtained by transduction with 4 factors (OSMK), and with 3factors without c-MYC plus miRNAs (OSK+). FIG. 4B shows the morphologyof ES-like colonies from a subset of the samples counted in FIG. 4A.

FIG. 5 shows expression of ES cell markers in iPS cells produced bynuclear reprogramming of mouse Tail Tip Fibroblasts (TTFs) with 4factors (OCT3/4, SOX2, c-MYC, and KLF4) and with 3 factors (OCT3/4,SOX2, and KLF4, i.e. “Myc(−)3f”)+mmu-miR-295/295* or DsRed.

FIGS. 6A-B show the results of MEFs infected with 3 factors (Oct3/4,c-MycWT, and Klf4, i.e., “Sox(−)”) with mmu-miR-290-295 cluster,290-5p/290-3p(mmu-miR-290), 291a-5p/291a-3p(mmu-miR-291a),292-5p/292-3p(mmu-miR-292), 293/293*(mmu-miR-293), 294/294*(mmu-miR-294)or 295/295*(mmu-miR-295). FIG. 6A shows the number of Nanog GFP positivecolonies. FIG. 6B shows expression of ES marker genes in iPS cellschecked with RT-PCR.

FIGS. 7A-C show the results of iPS induction with Fb-Ng MEFs (MEFsderived from Fbx 15-β geo/Nanog-IRES-Puro^(r) reporter mouse)over-expressing Oct3/4, c-Myc, and Klf4 (“Sox(−)”)+mmu-miR-295/295* orhsa-miR-302-367 cluster miRNAs. FIG. 7A shows cell morphology of MEFstransduced with Oct3/4, c-Myc, and Klf4 (“Sox(−)”)+mmu-miR-295/295*.FIG. 7B shows chimeras derived from iPS cells induced withSox(−)3f+mmu-miR-295/295*. FIG. 7C shows embryoid body (EB)-mediated invitro differentiation by human iPS cells. Human iPS cells (61B1, 61N2)were established by transduction of 4 genes (OCT3/4, KLF4, SOX2, andc-MYC, i.e., “OSMK”) or 3 genes (OCT3/4, KLF4, and c-MYC, i.e.,“OMK(SOX(−)”) in the presence of hsa-miR-302-367 cluster miRNA. Afterculturing for 16 days, immunohistochemistry analysis was performed inthe cells by using an antibody against each of a-fetoprotein (AFP) whichis a differentiation marker for endodermal cells, α-smooth muscle actin(a-SMA) which is a differentiation marker for mesodermal cells, and GFAP(DAKO) which is a differentiation marker for ectodermal cells. Nucleiwere stained with Hoechst 33342 (Invitrogen).

FIG. 8 shows cell morphology of iPS cells induced with OSMK; SOX(−)(OKM)+hsa-miR-302-367 cluster miRNA; and OCT3/4+KLF4+hsa-miR-302-367cluster miRNA.

TABLE 3 shows iPS induction by transduction with 4 factors (OCT3/4,SOX2, MYC, KLF4, i.e., “OSMK”), with 3 factors (SOX(−)3factors, i.e.,“OMK”) plus various miRNAs (OMK:mock or miRNAs=2.5:1.5), and with 2factors (OCT3/4+KLF4, i.e., “OK”) plus various miRNAs. Transduction withDsRed was performed as a as control. On Day 40 after infection, thenumber of ES-like colonies was counted. TABLE 3 shows the results of sixindependent experiments (Exp. 54, 61, 63, 114, 130 and 133).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for efficiently preparing inducedpluripotent stem cells through reprogramming of one or more somaticcells. In particular, the present invention provides efficientpreparation of induced pluripotent stem cells in the presence of miRNA.The present invention also provides efficient preparation with orwithout using a suspected tumorigenic factor: c-Myc. The presentinvention also provides efficient preparation with or without usingSox2. The nuclear reprogramming is preferably performed without c-Mycand/or Sox2.

The inventors of the present invention have conducted intensive studies,and as a result, they have found that induced pluripotent stem cells canbe efficiently prepared by introduction of nuclearreprogramming-inducing gene(s) into somatic cells in the presence ofspecific miRNA. The present invention was achieved on the basis of theabove findings.

The present invention thus provides a method of preparing inducedpluripotent stem cells, comprising nuclear reprogramming with a nuclearreprogramming factor in the presence of miRNA, wherein said miRNA has aproperty of providing a higher nuclear reprogramming efficiency in thepresence of said miRNA than in the absence thereof.

A preferred embodiment of the present invention provides theaforementioned method wherein: (a) said miRNA is expressed in embryonicstem cells at a higher level than in somatic cells; and/or (b) saidmiRNA has a property of providing a higher nuclear reprogrammingefficiency in the presence of said miRNA than in the absence thereofand/or (c) said nuclear reprogramming is performed in the presence ofincreased levels of one or more miRNAs as compared to the level(s)present in the somatic cell prior to nuclear reprogramming.

Another preferred embodiment of the present invention provides: theaforementioned method wherein the nuclear reprogramming factor is eithera single substance, or a combination of a plurality of substances, whichis/are positive in the screening method of nuclear reprogramming factordescribed in International Publication No. WO2005/80598 A1, incorporatedby reference herein in its entirety; the aforementioned method whereinthe nuclear reprogramming factor is either a gene product of a singlegene, or a combination of gene products of a plurality of genes, whichis/are positive in the screening method of nuclear reprogramming factordescribed in International Publication No. WO2005/80598 A1, incorporatedby reference herein in its entirety; the aforementioned method whereinthe nuclear reprogramming with the nuclear reprogramming factor iscarried out by introduction of the aforementioned gene(s) and/orsubstance(s) into somatic cells; the aforementioned method whereinintroduction of the aforementioned gene(s) into somatic cells is carriedout with a recombinant vector; and the aforementioned method wherein thenuclear reprogramming with the nuclear reprogramming factor is carriedout by introduction of gene product(s) of the aforementioned gene(s)into somatic cells.

Yet another preferred embodiment of the present invention provides theaforementioned method wherein: the gene encoding the reprogrammingfactor comprises one or more gene(s) selected from an Oct family gene, aKlf family gene, a Sox family gene, a Myc family gene, a Lin familygene, and a Nanog gene, preferably a combination of two genes selectedfrom the aforementioned genes except for the Myc family genes of Soxfamily genes, more preferably a combination of three genes, andparticularly preferably a combination four or more genes. In a preferredembodiment, the combination can be any combination of factors which doesnot comprise c-Myc or Sox2.

More preferable combinations are: (a) a combination of two genescomprising of an Oct family gene and a Sox family gene; (b) acombination of three genes comprising an Oct family gene, a Klf familygene, and a Sox family gene; and (c) a combination of four genescomprising an Oct family gene, a Sox family gene, a Lin family gene, anda Nanog gene. Further, it is also preferable to combine any of the abovegenes with a TERT gene and/or a SV40 Large T antigen gene. It may bepreferable to omit Klf family genes depending on the situation. The Mycfamily genes may or may not be included in these combinations.Combinations without the Myc family gene can be suitably used accordingto the present invention.

Among these embodiments, particularly preferable combinations are: acombination of two genes comprising Oct3/4 and Sox2; a combination ofthree genes comprising Oct3/4, Klf4, and Sox2; and a combination of fourgenes comprising Oct3/4, Sox2, Lin28, and Nanog. It is also preferableto combine any of the above genes with a TERT gene and/or a SV40 Large Tantigen gene. It may be preferable to omit Klf4 depending on thesituation. c-Myc may be included in these combinations. However,combinations without c-Myc can be suitably used in the presentinvention.

Other preferable combinations are: (a) a combination of two genescomprising of an Oct family gene and a Klf family gene; (b) acombination of three genes comprising an Oct family gene, a Klf familygene, and a Myc family gene. Yet another preferred embodiment of thepresent invention provides: the aforementioned method wherein thesomatic cells are those derived from mammals including human, mouse,rat, cattle, sheep, horse, monkey, and hamster, preferably somatic cellsfrom human or mouse, and most preferably somatic cells from human; theaforementioned method wherein the somatic cells are human embryoniccells, or adult human-derived somatic cells; and the aforementionedmethod wherein the somatic cells are somatic cells collected from apatient.

Yet another preferred embodiment of the present invention provides theaforementioned method wherein the miRNA comprises one or more miRNA(s)included in the RNA sequences specified by the registration names of themiRBase database or the accession numbers shown in Table 1 or Table 2;the aforementioned method wherein the RNA sequences specified by theregistration names of the miRBase database (and the accession numbers)shown in Table 1 or Table 2 comprise one or more RNA(s) selected fromhsa-miR-372 (MI0000780), hsa-miR-373 (MI0000781), hsa-miR-302b(MI0000772), hsa-miR-302c (MI0000773), hsa-miR-302a (MI0000738),hsa-miR-302d (MI0000774), hsa-miR-367 (MI0000775), hsa-miR-520c(MI0003158), mmu-miR-290 (MI0000388), mmu-miR-291a (MI0000389),mmu-miR-294 (MI0000392), and mmu-miR-295 (MI0000393); the aforementionedmethod wherein the miRNA comprises miRNA included in RNA specified byhsa-miR-302-367; and the aforementioned method wherein the miRNAcomprises one or more miRNA(s) included in one or more RNA sequence(s)selected from the RNA sequences represented by SEQ IDS: 1 to 14 in theSequence Listing.

The present invention provides an oligonucleotide comprising at least 10contiguous nucleotides in the nucleotide sequence of the miRNA of thepresent invention, and an antisense oligonucleotide having a sequencethat is complementary to that of the above oligonucleotide. The presentinvention also provides an oligonucleotide comprising at least 15, atleast 20, at least 30, at least 50, or at least 60 contiguousnucleotides in the nucleotide sequence of the miRNA of the presentinvention. The present invention also provides an oligonucleotidecomprising at least 70, at least 80, at least 100, at least 150, atleast 200, at least 300, at least 400, at least 600, or at least 800contiguous nucleotides in the nucleotide sequence of the miRNA of thepresent invention.

The present invention also provides induced pluripotent stem cells thatcan be obtained by the aforementioned method. In addition, the presentinvention also provides somatic cells obtained by inducingdifferentiation from the abovementioned induced pluripotent stem cells.

Further, the present invention provides a stem cell therapy comprisingtransplanting somatic cells into a patient, wherein the somatic cellsare obtained by inducing differentiation from induced pluripotent stemcells that are obtained according to the aforementioned method by usingsomatic cells isolated and collected from a patient.

In addition, the present invention provides a method for evaluation ofphysiological effect or toxicity of a compound, a drug, or a toxicagent, with use of various cells obtained by inducing differentiationfrom induced pluripotent stem cells that are obtained by theaforementioned method.

Further, the present invention provides: a method for preparing inducedpluripotent stem cells which uses miRNA expressed in embryonic stemcells at a higher level than in somatic cells, and having a property ofproviding a higher nuclear reprogramming efficiency in the presence ofsaid miRNA than in the absence thereof; and a nuclear reprogrammingmethod of somatic cells which uses miRNA expressed in embryonic stemcells at a higher level than in somatic cells, and having a property ofproviding a higher nuclear reprogramming efficiency in the presence ofsaid miRNA than in the absence thereof.

In addition, the present invention provides methods comprising the useof miRNA expressed in embryonic stem cells at a higher level than insomatic cells (e.g., the miRNA may be expressed at levels which arehigher in the ES cell as compared to the ES cell which hasdifferentiated or which has begun differentiating such as determined byRT-PCR or Northern blot analysis), and having a property of providing ahigher nuclear reprogramming efficiency in the presence of said miRNAthan in the absence thereof, for preparation of induced pluripotent stemcells; and methods relating to the use of miRNA expressed in embryonicstem cells at a higher level than in somatic cells, and having aproperty of providing a higher nuclear reprogramming efficiency in thepresence of the miRNA than in the absence thereof, for nuclearreprogramming of somatic cells. In other words, nuclear reprogramming,and thus, induced pluripotent stem cell production, can be performed inthe presence of miRNA and in the absence of miRNA. The nuclearreprogramming may also be performed in the presence of various amountsand/or kinds of miRNA, such that, for example, the efficiency of thenuclear reprogramming is increased when the level of the miRNA isincreased in the somatic cell prior to nuclear reprogramming.

In addition, the present invention provides methods comprising the useof miRNA having a property of providing a higher nuclear reprogrammingefficiency in the presence of said miRNA than in the absence thereof,for preparation of induced pluripotent stem cells. For example, thepresence of added miRNA can provide the formation of an inducedpluripotent stem cell as compared to the lack of formation in theabsence of the miRNA. Also, for example when nuclear reprogramming isperformed on the same number of somatic cells in the presence of anuclear reprogramming factor containing the same components in the sameconcentrations with and without addition of miRNA, increased efficiencycan be observed when a greater number of induced pluripotent stem cellsare generated in the sample which comprises the addition of miRNA thanin the sample without the addition of miRNA. In another embodiment,increased efficiency of induced pluripotent stems cell production canalso be achieved with increased amounts of miRNA as compared to miRNAamounts present in the somatic cell prior to nuclear reprogramming.

BEST MODE FOR CARRYING OUT THE INVENTION

The methods of the present invention relate to, e.g., a method forpreparing induced pluripotent stem cells, comprising nuclearreprogramming with a nuclear reprogramming factor in the presence ofmiRNA, wherein said miRNA has a property of providing a higher nuclearreprogramming efficiency in the presence of said miRNA than in theabsence thereof. In a preferred embodiment of the present invention, (a)said miRNA is expressed in embryonic stem cells at a higher level thanin somatic cells; and (b) said RNA has a property of providing a highernuclear reprogramming efficiency in the presence of said miRNA than inthe absence thereof.

As for the miRNA, for example, its classification and in vivo functionsare described in Jikken Igaku (Experimental Medicine), 24, pp. 814-819,2006; microRNA Jikken Purotokoru (microRNA Experimental Protocol), pp.20-35, 2008, YODOSHA CO., LTD. The number of nucleotides of miRNA is forexample 18 to 25, and preferably about 19 to 23. At present, a databasestoring data relating to about 1,000 miRNA sequences is available (forexample, miRBase, Griffiths-Jones et al. Nucleic Acids Research36:D154-D158, 2008 (published online Nov. 8, 2007), see alsohttp://microrna.sanger.ac.uk/sequences/index.shtml [online]), and it ispossible for those skilled in the art to obtain any miRNA datatherefrom, and to readily extract miRNA expressed in embryonic stemcells at a higher level than in somatic cells. In addition, it is alsopossible to readily specify miRNA expressed in embryonic stem cells at ahigher level than in somatic cells by confirming the difference in miRNAexpression between embryonic stem cells and somatic cells with use ofavailable techniques for those skilled in the art such as miRNAmicroarray and real-time PCR analyses.

The difference in the nuclear reprogramming efficiency with and withoutmiRNA can be understood by the following manner, as specificallydescribed in Examples of this application: transgenic mice are generatedby insertion of sequences encoding Enhanced Green Fluorescent Protein(EGFP) and a puromycin resistance gene downstream of a Nanog genepromoter region, the expression of which is specific to ES cells; then,three genes, for example, Oct3/4, Sox2, and Klf4, and various miRNAs areintroduced into embryonic fibroblasts derived from these transgenic miceto induce nuclear reprogramming, followed by confirmation of theproduction efficiency of induced pluripotent stem cells. The productionefficiency can be determined, for example, by counting the number ofcolonies. More specifically, the number of colonies can be compared bythe following manner: drug selection is started from the 21st day afterintroduction of the above genes and miRNA; and the number of totalcolonies and the number of Nanog GFP positive colonies (GFP, theexpression of which is induced by the Nanog gene promoter region, isobservable under fluorescent microscopy) are counted on the 28th day. Itshould be understood, however, that: the confirmation of the nuclearreprogramming efficiency is not limited to the above method; appropriatemodification and alteration can be made in the above method; and anyappropriate method can be employed by those skilled in the art.

As for the miRNA, it is preferable to use miRNA derived from the sameanimal species as the target animal whose somatic cells are to bereprogrammed. Usable miRNA includes wild type miRNA as well as miRNAs inwhich one to several nucleotides (for example 1 to 6 nucleotides,preferably 1 to 4 nucleotides, more preferably 1 to 3 nucleotides, yetmore preferably 1 or 2 nucleotides, and most preferably 1 nucleotide)are substituted, inserted, and/or deleted, and which are capable ofexerting equivalent functions to those of the wild type miRNA in vivo.For example, the miRNA of the present invention includes miRNAs in whichone to several nucleotides are substituted, inserted, and/or deleted,and which increase the efficiency of iPS cell production. The miRNA ofthe present invention also includes miRNAs in which one to severalnucleotides are substituted, inserted, and/or deleted, and which improvethe efficiency of nuclear reprogramming. The miRNA of the presentinvention also includes miRNAs in which one to several nucleotides aresubstituted, inserted, and/or deleted, and which regulate DNAmethylation. The present invention also includes such miRNAs wherein theDNA methylation is down-regulated. The present invention also includessuch miRNAs wherein the DNA methylation is de novo DNA methylation.

Examples of the miRNA preferably used in the methods of the presentinvention can include, but are not limited to, one or more miRNA(s)included in the following RNA sequences registered in the miRBase:hsa-miR-372 (MI0000780), hsa-miR-373 (MI0000781), hsa-miR-302b(MI0000772), hsa-miR-302c (MI0000773), hsa-miR-302a (MI0000738),hsa-miR-302d (MI0000774), hsa-miR-367 (MI0000775), hsa-miR-520c(MI0003158), mmu-miR-290 (MI0000388), mmu-miR-291a (MI0000389),mmu-miR-294 (MI0000392), and mmu-miR-295 (MI0000393) (Numbers in thebrackets respectively indicate miRBase accession numbers. The symbol“hsa-miR-” represents human miRNA, and the symbol “mmu-miR-” representsmouse miRNA.).

In the method of the present invention, miRNAs that have been confirmedto improve the nuclear reprogramming efficiency in the above manner canbe used either alone or in combinations of two or more types. Inaddition, a plurality of miRNAs forming a cluster may also be used. Forexample, hsa-miR-302-367 which is available as a miRNA cluster, orindividual miRNAs from the hsa-miR-302-367 cluster, and the like may beused. Examples of RNA sequences for use in the present invention areshown in SEQ IDS: 1 to 14 in the Sequence Listing. SEQ ID: 1:mmu-miR-294 (MI0000392); SEQ ID: 2: mmu-miR-295 (MI0000393); SEQ ID: 3:hsa-miR-372 (MI0000780); SEQ ID: 4: hsa-miR-373 (MI0000781); SEQ ID: 5:hsa-miR-302b (MI0000772); SEQ ID: 6: hsa-miR-302c (MI0000773); SEQ ID:7: hsa-miR-302a (MI0000738); SEQ ID: 8: hsa-miR-302d (MI0000774); SEQID: 9: hsa-miR-367 (MI0000775); SEQ ID: 10: hsa-miR-520c (MI0003158);SEQ ID: 11: mmu-miR-291a (MI0000389); SEQ ID:13: mmu-miR-290(MI0000388), and SEQ ID:14: hsa-miR-371-373 cluster. In addition, RNArepresented by SEQ ID: 12: hsa-miR-302-367 cluster can also bepreferably used. Among these RNA sequences, some RNA sequences mayinclude a plurality of miRNAs within one sequence. Use of such an RNAsequence may achieve efficient production of iPS cells. Further, an RNAsequence including a plurality of miRNAs within one sequence and one ormore other RNA sequence(s) including one or more miRNA(s) can also beused in combination.

miRNA is non-coding RNA which is not translated into a protein. miRNA isfirst transcribed as pri-miRNA from a corresponding gene, then thispri-miRNA generates pre-miRNA having a characteristic hairpin structureof about 70 nucleotides, and this pre-miRNA is further processed intomature miRNA, which is mediated by Dicer. In the present invention, notonly mature miRNA but also pri-miRNA or pre-miRNA can be used as long asthe effect of the present invention is not impaired. In addition, miRNAfor use in the present invention may be either natural type ornon-natural type. Thus, any small RNA or RNA precursor may be used aslong as the effect of the pre/sent invention is not impaired.

The production method of miRNA for use in the present invention is notspecifically limited, although the production can be achieved, forexample, by a chemical synthetic method or a method using geneticrecombination technique. When the production is carried out by a methodusing genetic recombination technique, miRNA for use in the presentinvention can, for example, be produced through transcription reactionwith use of a DNA template and a RNA polymerase obtained by means ofgene recombination. Examples of usable RNA polymerase include a T7 RNApolymerase, a T3 RNA polymerase, and a SP6 RNA polymerase.

Alternatively, a recombinant vector capable of expressing miRNA can beproduced by insertion of miRNA-encoding DNA into an appropriate vectorunder the regulation of expression control sequences (promoter andenhancer sequences and the like). The type of vector used herein is notspecifically limited, although DNA vectors are preferred. Examplesthereof can include viral vectors and plasmid vectors. The viral vectoris not specifically limited, although retroviral vectors, adenoviralvectors, adeno-associated viralvectors, and the like can be employed. Inaddition, as to the above plasmids, mammalian expression plasmids wellknown to those skilled in the art can be employed.

Methods for using a retrovirus as a vector are disclosed in WO2007/69666 A1; Takahashi et al., Cell 126:663-676, 2006; and Takahashiet al., Cell 131:861-872, 2007, which are herein incorportated byreference in their entireties. Methods for using a lentivirus as avector are disclosed in Yu et al., Science 318:1917-1920, 2007, which isherein incorporated by reference in its entirety. Methods for usingadenovirus as a vector are disclosed in Stadtfeld et al., Science322:945-949, 2008, which is herein incorporated by reference in itsentirety. Methods for using a plasmid as a non-viral vector aredisclosed in U.S. Provisional Application No. 61/071,508; U.S.Provisional Application No. 61/136,246; U.S. Provisional Application No.61/136,615; and U.S. Provisional application Ser. No. ______ (AttorneyDocket No. V35667) entitled “Method for Nuclear Reprogramming” filedNov. 21, 2008; and Okita et al., Science 322:949-953, 2008, which areherein incorporated by reference in their entireties. One of ordinaryskill in the art could choose and use an appropriate method from amongthe above known methods, or from any of the other known methods orvectors available in the prior art.

Nuclear reprogramming can be performed in the presence of miRNA in anynumber of ways. The manner of providing the miRNA is not specificallylimited, although examples thereof can include a method for directlyinjecting miRNA into nuclei of somatic cells, and a method forintroducing an appropriate recombinant vector capable of expressingmiRNA into somatic cells. However, these methods are not to beconsidered as limiting.

The method for introducing a recombinant vector into somatic cells isnot specifically limited, and can be carried out by any method wellknown to those skilled in the art. Examples of the employable methodscan include transient transfection, microinjection, a calcium phosphateprecipitation method, liposome-mediated transfection, DEAEdextran-mediated transfection, electroporation, and methods comprisingthe use of a gene gun.

As to confirming a nuclear reprogramming factor, for example, thescreening method of nuclear reprogramming factor described inInternational Publication No. WO2005/80598 A1, incorporated by referenceherein in its entirety, can be used. Those skilled in the art are ableto screen a nuclear reprogramming factor for use in the method of thepresent invention by referring to the above publication. In addition,the nuclear reprogramming factor can also be confirmed by using a methodin which appropriate modification or alteration has been made in theabove screening method.

Examples of the combination of genes encoding reprogramming factors aredisclosed in International Publication No. WO2007/069666 A1 and itsfamily member U.S. patent application Ser. No. 12/213,035 and U.S.patent application Ser. No. 12/289,873, filed Nov. 6, 2008, entitled“Nuclear Reprogramming Factor and Induced Pluripotent Stem Cells” whichare incorporated by reference herein in their entireties. Those skilledin the art are able to appropriately select a gene that can bepreferably used for the method of the present invention by referring tothe above publication. In addition, other examples of the combinationsof genes encoding reprogramming factors are disclosed, for example, inYu et al., Science 318:1917-20, 2007, incorporated by reference hereinin its entirety. Accordingly, those skilled in the art are able tounderstand the variety of the combination of genes encodingreprogramming factors, and are able to employ an appropriate combinationof genes in the method of the present invention, which combination isnot disclosed in International Publication No. WO2007/069666 A1 or Yu etal., Science 318:1917-20, 2007, by using the screening method of nuclearreprogramming factor described in International Publication No.WO2005/80598 A1.

Examples of the gene encoding a reprogramming factor that can be usedfor the method of the present invention can include: one or more gene(s)selected from an Oct family gene, a Klf family gene, a Sox family gene,a Myc family gene, a Lin family gene, and a Nanog gene; preferably oneor more gene(s) selected from an Oct family gene, a Klf family gene, aSox family gene, a Lin family gene, and a Nanog gene, and excluding aMyc family gene; one or more gene(s) selected from an Oct family gene, aKlf family gene, a Myc family gene, a Lin family gene, and a Nanog gene,and excluding a Sox family gene; more preferably a combination of twogenes; yet more preferably a combination of three genes; and mostpreferably a combination of four genes.

Regarding the Oct family gene, Klf family gene, Sox family gene, and Mycfamily gene, specific examples of these family genes are described inInternational Publication No. WO2007/069666 A1. Regarding the Lin familygene, those skilled in the art are able to extract the family gene in asimilar way. Examples of the Lin family genes include, for example,Lin28 and Lin28b. The NCBI accession numbers of Lin28 are NM 145833(mouse) and NM_(—)024674 (human). The NCBI accession numbers of Lin28bare NM_(—)001031772 (mouse) and NM_(—)001004317 (human).

In addition, reprogramming factor(s) encoded by one or more gene(s)selected from an Oct family gene, a Klf family gene, a Sox family gene,a Myc family gene, a Lin family gene, and a Nanog gene, may besubstituted by, for example a cytokine, or one or more other lowmolecular weight compound(s) in some cases. Examples of such lowmolecular weight compound(s) can include low molecular weight compoundshaving an enhancing action on the expression of one or more gene(s)selected from an Oct family gene, a Klf family gene, a Sox family gene,a Myc family gene, a Lin family gene, and a Nanog gene. Those skilled inthe art are able to readily screen such low molecular weightcompound(s).

More preferable combinations of genes are as follows:

(a) a combination of two genes comprising an Oct family gene and a Soxfamily gene;(b) a combination of three genes comprising an Oct family gene, a Klffamily gene, and a Sox family gene;(c) a combination of four genes comprising an Oct family gene, a Soxfamily gene, a Lin family gene, and a Nanog gene;(d) a combination of two genes comprising an Oct family gene and a Klffamily gene; and(e) a combination of three genes comprising an Oct family gene, a Klffamily gene, and a Myc family gene.However, these combinations are not to be considered as limiting.

All of these genes are commonly present in mammals, including human. Inorder to use the above genes according to the present invention, genesderived from any mammal (for example, derived from a mammal such ashuman, mouse, rat, cattle, sheep, horse, and monkey) can be employed. Inaddition, it is also possible to use a wild type gene product, as wellas mutant gene products in which several amino acids (for example 1 to10 amino acids, preferably 1 to 6 amino acids, more preferably 1 to 4amino acids, yet more preferably 1 to 3 amino acids, and most preferably1 or 2 amino acids) have been substituted, inserted, and/or deleted, andwhich have comparable equivalent functions to those of the wild typegene product. For example, as to the c-Myc gene product, a stable typevariant, e.g., (T58A) and the like may also be used as well as the wildtype. The same principle can be applied to other gene products.

In addition to the above genes, a gene encoding a factor which inducesimmortalization of cells may also be combined. As disclosed inInternational Publication No. WO2007/069666 A1, for example, one or moregene(s) selected from a TERT gene, and following genes: SV40 Large Tantigen, HPV16 E6, HPV16 E7, and Bmil, can be either solely used orjointly used in an appropriate combination.

Preferable combinations are as follows, for example:

(e) a combination of four genes comprising an Oct family gene, a Klffamily gene, a Sox family gene, and a TERT gene;(f) a combination of four genes comprising an Oct family gene, a Klffamily gene, a Sox family gene, and a SV40 Large T antigen gene; and(g) a combination of five genes comprising an Oct family gene, a Klffamily gene, a Sox family gene, a TERT gene, and a SV40 Large T antigengene.The Klf family gene may be omitted from the above combinations.

Further, in addition to the above genes, one or more gene(s) selectedfrom Fbx15, ERas, ECAT15-2, Tell, and β-catenin may be combined, and/orone or more gene(s) selected from ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3,Sox15, ECAT15-1, Fthl17, Sal14, Rex1, UTF1, Stella, Stat3, and Grb2 mayalso be combined. These combinations are specifically described inInternational Publication No. WO2007/069666 A1.

Particularly preferable combinations of genes are as follows:

(1) a combination of two genes comprising Oct3/4 and Sox2;(2) a combination of three genes comprising Oct3/4, Klf4, and Sox2;(3) a combination of four genes comprising Oct3/4, Sox2, Lin28, andNanog;(4) a combination of four genes comprising Oct3/4, Sox2, TERT, and SV40Large T antigen gene;(5) a combination of five genes comprising Oct3/4, Klf4, Sox2, TERT, andSV40 Large T antigen gene;(6) a combination of two genes comprising Oct3/4 and Klf4;(7) a combination of three genes comprising Oct3/4, Klf4, and c-Myc; and(8) a combination of four genes comprising Oct3/4, Sox2, Klf4, andc-Myc.However, these combinations are not to be considered as limiting.

The factors including the gene products as mentioned above may also becombined with one or more gene product(s) of gene(s) selected from:Fbx15, Nanog, ERas, ECAT15-2, Tell, and β-catenin. Further, thesefactors may also be combined with one or more gene product(s) of gene(s)selected from: ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, ECAT15-1,Fthl17, Sal14, Rex1, UTF1, Stella, Stat3, and Grb2, for example. Thesegene products are disclosed in International Publication No.WO2007/069666 A1. However, gene products that can be included in thenuclear reprogramming factors of the present invention are not limitedto the gene products of genes specifically described above. The nuclearreprogramming factors of the present invention can include other geneproducts which can function as a nuclear reprogramming factor, as wellas one or more factors involving differentiation, development, orproliferation, and factors having other physiological activities. Itshould be understood that the aforementioned aspect may also be includedwithin the scope of the present invention.

Among these genes, if one or more gene product(s) is/are alreadyexpressed in somatic cells to be reprogrammed, such gene products can beexcluded from the factors to be introduced. For example, one or moregene(s) besides the already-expressed gene(s) can be introduced intosomatic cells by an appropriate gene introduction method, for example, amethod using a recombinant vector. Alternatively, among these genes, ifone or more gene product(s) is/are introduced into nuclei by a techniquesuch as addition of an HIV virus-derived TAT peptide and/or nuclearlocalization signal to form a fusion protein or by a technique such asnuclear microinjection, or simply by addition of a small moleculecapable of diffusing across the plasma membrane, the other one or moregene(s) can be introduced by an appropriate gene introduction method,for example, a method using a recombinant vector.

In addition, a gene product serving as a nuclear reprogramming factormay be either a protein itself produced from the abovementioned gene, orin the form of a fusion gene product between such a protein and anotherprotein, a peptide, or the like. For example, a fusion protein havingGreen Fluorescent Protein (GFP) and a fusion gene product having apeptide such as a histidine tag may also be used. Further, use of aprepared fusion protein having a HIV virus-derived TAT peptide enablesthe promotion of endocytosis of a nuclear reprogramming factor throughcell membrane, and also enables the induction of reprogramming by simplyadding such a fusion protein into the medium while avoiding complicatedmanipulations such as gene introduction. The preparation method of theaforementioned fusion gene product is well known to those skilled in theart, and therefore those skilled in the art are able to readily designand prepare an appropriate fusion gene product according to the purpose.

In this application, the term “induced pluripotent stem cells (iPScells)” refers to cells having similar properties to those of ES cells,and more specifically the term includes undifferentiated cells which arereprogrammed from somatic cells and have pluripotency and proliferationpotency. However, this term is not to be construed as limiting in anysense, and should be construed to have its broadest meaning. Thepreparation method of induced pluripotent stem cells with the use of anuclear reprogramming factor is described in International PublicationNo. WO2005/80598 A1 (the term “ES-like cell” is used in thispublication), and methods for isolating induced pluripotent stem cellsare also specifically described. In addition, specific examples of thereprogramming factor and specific examples of the reprogramming methodof somatic cells with use of such a reprogramming factor are disclosedin International Publication No. WO2007/069666 μl. Accordingly, it isdesirable for those skilled in the art to refer to these publicationsfor carrying out the present invention.

The preparation method of induced pluripotent stem cells from somaticcells by the method of the present invention is not specificallylimited, and any method can be employed as long as the method enablesnuclear reprogramming of somatic cells with a nuclear reprogrammingfactor in the presence of miRNA in an environment where somatic cellsand induced pluripotent stem cells can grow. For example, a vectorcomprising a gene which can express a nuclear reprogramming factor canbe used to introduce such a gene into somatic cells, and at either thesame or different timing, a recombinant vector which can express miRNAcan be introduced into the somatic cells. If such vectors are used, twoor more genes may be incorporated into a vector to effect simultaneousexpression of respective gene products in somatic cells.

When gene(s) and/or miRNA are introduced into somatic cells with use ofa vector which can express the above gene(s), the expression vector maybe introduced into somatic cells that have been cultured on feedercells, or the expression vector may also be introduced into somaticcells alone. The latter method is sometimes more suitable in order toimprove the introduction efficiency of the expression vector. As to thefeeder cells, there may be appropriately used feeder cells for use inculture of embryonic stem cells. Examples thereof can include primaryculture cells of 14 or 15 day-mouse embryonic fibroblasts and STO cellsof fibroblast cell line, which are treated with either radiation or adrug such as mitomycin C.

The culture of somatic cells introduced with a nuclear reprogrammingfactor under an appropriate condition leads to autonomous nuclearreprogramming, as a result of which induced pluripotent stem cells canbe produced from somatic cells. The process for introducing a geneencoding a nuclear reprogramming factor and/or miRNA into somatic cellswith use of an expression vector to thereby obtain induced pluripotentstem cells can be performed in accordance with, for example, a methodusing a retrovirus. Examples of such method include methods described inpublications such as Takahashi et al., Cell 126:663-76, 2006; Takahashiet al., Cell 131:861-72, 2007; Yu et al., Science 318:1917-20, 2007.When human induced pluripotent stem cells are to be produced, it isdesirable to set the cell culture density after the introduction of anexpression vector to be lower than normal cases for culturing animalcells. For example, it is preferable to keep culturing at a density of1×10⁴ to 1×10⁵ cells/10 cm dish, and more preferably about 5×10⁴cells/10 cm dish. The medium for use in culture is not specificallylimited, and can be appropriately selected by those skilled in the art,although for example it is sometimes preferable to use a medium suitablefor human ES cell culture for the production of human inducedpluripotent stem cells. The medium selection and culture condition canbe referred to the above publications.

Thus produced induced pluripotent stem cells can be checked with variousmarkers specific to undifferentiated cells, and the means therefor isdescribed in the above publications specifically in detail. For example,some pluripotent cell markers include: alkaline phosphatase (AP); ABCG2;stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60;TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; βIII-tubulin; α-smoothmuscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1;zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cellassociated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7;ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14;undifferentiated embryonic cell transcription factor (Utf1); Rex1; p53;G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a;Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4;Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3;CYP25A1; developmental pluripotency-associated 2 (DPPA2); and T-celllymphoma breakpoint 1 (Tell); DPPA3/Stella; DPPA4. Other markers caninclude Dnmt3L; Sox15; Stat3; Grb2; SV40 Large T Antigen; HPV16 E6;HPV16 E7, 13-catenin, and Bmi1. Such cells can also be characterized bythe down-regulation of markers characteristic of the differentiated cellfrom which the iPS cell is induced. For example, iPS cells derived fromfibroblasts may be characterized by down-regulation of the fibroblastcell marker Thy1 and/or up-regulation of SSEA-3 and 4. It is understoodthat the present invention is not limited to those markers listedherein, and encompasses markers such as cell surface markers, antigens,and other gene products including ESTs, RNA (including microRNAs andantisense RNA), DNA (including genes and cDNAs), and portions thereof.

Various media capable of retaining undifferentiation property andpluripotency of ES cells and various media incapable of retaining theseproperties are known in the art, and appropriate combination of thesemedia enables efficient isolation of induced pluripotent stem cells. Thedifferentiation ability and proliferation potency of thus isolatedinduced pluripotent stem cells can be readily checked by those skilledin the art, with use of general checking means for ES cells. Inaddition, colonies of induced pluripotent stem cells can be obtained bygrowing thus produced induced pluripotent stem cells under anappropriate condition, and the presence of these induced pluripotentstem cells can be identified with reference to the shape of theircolonies. For example, it is known that mouse induced pluripotent stemcells form raised colonies, while human induced pluripotent stem cellsform flat colonies. These colony shapes are respectively very similar tothose of mouse ES cells and human ES cells, and those skilled in the artare thus able to identify these produced induced pluripotent stem cellswith reference to the shape of their colonies.

The type of somatic cell to be reprogrammed by the method of the presentinvention is not specifically limited, and any somatic cell can be used.For example, somatic cells derived from any mammal (for example, derivedfrom a mammal such as human, mouse, rat, cattle, sheep, horse, andmonkey) can be employed. Not only embryonic somatic cells but alsoneonatal somatic cells, matured somatic cells, and tissue stem cells mayalso be used. In addition, various somatic cells such as skin cells,liver cells, and gastric mucosa cells can be reprogrammed. For use ofinduced pluripotent stem cells in therapies against diseases, it isdesirable to use somatic cells isolated from the patient. For example,somatic cells involved in a disease and somatic cells associated with atherapy for a disease can be used.

The application of induced pluripotent stem cells produced by the methodof the present invention is not specifically limited, and these cellscan be used for every examination/study to be performed with use of EScells, and for any disease therapy which utilizes ES cells. For example,induced pluripotent stem cells obtained by the method of the presentinvention can be induced into desired differentiated cells (such asnerve cells, myocardial cells, blood cells and insulin-producing cells)by treatment with retinoic acid, a growth factor such as EGF, orglucocorticoid, so that appropriate tissue can be formed. Stem celltherapies through autologous cell transplantation can be achieved byreturning these differentiated cells or tissue obtained in the abovemanner, into the patient. However, the application of the inducedpluripotent stem cells of the present invention is not to be limited tothe abovementioned specific aspects.

EXAMPLES

The present invention will be explained more specifically with referenceto the following examples. However, the scope of the present inventionis not limited to these examples.

Example 1 Preparation of Induced Pluripotent Stem Cells Through NuclearReprogramming of Mouse Embryonic Fibroblasts

pMXs-based retroviral vectors, which respectively encode each of threegenes of mouse-derived Oct3/4, Sox2, and Klf4, control DsRed or eachmiRNA of 18 types of miRNAs, were transfected into PLAT-E cells usingFuGENE 6 reagent (Roche) to get retroviruses. On the next day, embryonicfibroblasts (Nanog GFP MEF, WO2007/069666 A1) derived from transgenicmice generated by insertion of sequences encoding EGFP gene andpuromycin resistance gene downstream of a Nanog gene promoter region,were seeded at 1×10⁵ cells/well in 6-well plates. On the next day, thesecells were infected with retroviruses expressing Oct3/4, Sox2, Klf4, andeach type of miRNA selected from 18 types of miRNAs, at a ratio of 1 mlof virus mixture expressing these three factors to 1 ml of virussolution expressing miRNA or DsRed, so as to prepare induced pluripotentstem cells through nuclear reprogramming.

TABLE 1 miRNA sequence (other name(s) miRBase miRNA number indicated inparentheses) accession number 1 mmu-miR-150 MI0000172 2 mmu-miR-182MI0000224 3 mmu-miR-126 MI0000153 4 mmu-miR-290-295 cluster 5mmu-miR-290 MI0000388 (mmu-miR-290-5p/290-3p) 6 mmu-miR-291a MI0000389(mmu-miR-291a-5p/291a-3p) 7 mmu-miR-292 MI0000390(mmu-miR-292-5p/292-3p) 8 mmu-miR-294 MI0000392 (mmu-miR-294/294*) X(9)mmu-miR-295 MI0000393 (mmu-miR-295/295*) 10 mmu-miR-17-92 cluster 11mmu-miR-323 MI0000592 12 mmu-miR-130b MI0000408 13 mmu-miR-7a-1MI0000728 14 mmu-miR-7a-2 MI0000729 15 mmu-miR-205 MI0000248 16mmu-miR-200a MI0000554 17 mmu-miR-200c MI0000694 18 mmu-miR-mix*indicates star form of miRNA.

From the third day after infection, the cells were cultured in an EScell medium containing LIF. On the fourth day after infection, the cellswere harvested by trypsinization, and the whole amount thereof wasspread over mytomicin-C treated STO cells as feeder cells. Every otherday thereafter, the ES cell medium containing LIF was replaced. From the21st day after infection, drug selection was started with addition ofpuromycin at a final concentration of 1.5 μg/ml. On the 28th day, thenumber of Nanog GFP positive colonies (GFP, the expression of which isinduced by a Nanog gene promoter region, can be observed with the use offluorescent microscopy) was counted. As a control, DsRed was used inplace of miRNA. The results are shown in FIG. 1. It was found thatmmu-miR-294 and mmu-miR-295 could respectively improve the nuclearreprogramming efficiency when introduced into mouse embryonicfibroblasts together with three factors of Oct3/4, Sox2, and Klf4, andcould enable efficient establishment of induced pluripotent stem cells.

Example 2 Preparation of Induced Pluripotent Stem Cells Through NuclearReprogramming of Mouse Tail Tip Fibroblasts

pMXs-based retroviral vectors, which respectively encode each of threegenes of mouse-derived Oct3/4, Sox2, and Klf4, DsRed (control), ormmu-miR-295, were transfected into PLAT-E cells using FuGENE 6 reagent(Roche) to get retroviruses. On the next day, tail tip fibroblasts(Nanog GFP tailtip fibroblasts) derived from transgenic mice generatedby insertion of sequences encoding EGFP gene and puromycin resistancegene downstream of a Nanog gene promoter region, were seeded at 1×10⁵cells/well in 6-well plates. On the next day, these cells were infectedwith retroviruses expressing three factors of Oct3/4, Sox2, and Klf4,and either DsRed or mmu-miR-295, at a ratio of 1:1:1:1, so as to prepareinduced pluripotent stem cells through nuclear reprogramming.

Since the third day after infection, the cells were cultured in an EScell medium containing LIF. On the fourth day after infection, the cellswere harvested by trypsinization and the whole amount thereof was spreadover mytomicin-C treated STO cells as feeder cells. Every other daythereafter, the ES cell medium containing LIF was replaced. From the7th, 21st, or 28th day after infection, drug selection was started withaddition of puromycin at a final concentration of 1.5 μg/ml. On the 39thday, the number of total colonies and the number of Nanog GFP positivecolonies (GFP, the expression of which is induced by Nanog promoterregion, can be observed with fluorescent microscopy) were counted. Theresults are shown in FIG. 2. It was found that mmu-miR-295 could improvethe nuclear reprogramming efficiency when introduced into mouse tail tipfibroblasts together with three factors of Oct3/4, Sox2, and Klf4, andcould accelerate the reprogramming speed and enable efficientestablishment of induced pluripotent stem cells.

Example 3 Preparation of Induced Pluripotent Stem Cells Through NuclearReprogramming of Adult Human Dermal Fibroblasts

pMXs-based retroviral vectors, which encode three genes of human-derivedOCT3/4, SOX2, and KLF4, and control DsRed or either 23 types of miRNAsor an miRNA cluster, were transfected into PLAT-E cells using FuGENE 6reagent (Roche) to get retroviruses. On the next day, adult human dermalfibroblasts (aHDF) which were generated to express a rodent ecotropicvirus receptor Slc7a1 (aHDF-Slc7a1), were seeded at 3×10⁵ cells/well in6-cm dishes. On the next day, the cells were infected with retrovirusesexpressing three genes of OCT3/4, SOX2, KLF4, and various types ofmiRNAs, at a ratio of 1:1:1:1, so as to produce induced pluripotent stemcells through nuclear reprogramming.

TABLE 2 miRNA sequence (other miRNA name(s) indicated in miRBase numberparentheses) accession number 1 hsa-miR-371 MI0000779(hsa-miR-371-5p/371-3p) 2 hsa-miR-372 MI0000780 3 hsa-miR-373 MI0000781(hsa-miR-373/373*) 4 hsa-miR-371-373 cluster 5 hsa-miR-93 MI0000095(hsa-miR-93/93*) 6 hsa-miR-302a MI0000738 (hsa-miR-302a/302a*) 7hsa-miR-302b MI0000772 (hsa-miR-302b/302b*) 8 hsa-miR-302c MI0000773(hsa-miR-302c/302c*) 9 hsa-miR-302d MI0000774 (hsa-miR-302d/302d*) 10hsa-miR-367 MI0000775 (hsa-miR-367/367*) 11 hsa-miR-302-367 cluster 12hsa-miR-520a MI0003149 (hsa-miR-520a-5p/520a-3p) 13 hsa-miR-520bMI0003155 14 hsa-miR-520c MI0003158 (hsa-miR-520c-5p/520c-3p) 15hsa-miR-520d MI0003164 (hsa-miR-520d-5p/520d-3p) 16 hsa-miR-520eMI0003143 17 mmu-miR-290-295 cluster 18 mmu-miR-290 MI0000388(mmu-miR-290-5p/290-3p) 19 mmu-miR-291a MI0000389(mmu-miR-291a-5p/291a-3p) 20 mmu-miR-292 MI0000390(mmu-miR-292-5p/292-3p) 21 mmu-miR-293 MI0000391 (mmu-miR-293/293*) 22mmu-miR-294 MI0000392 (mmu-miR-294/294*) 23 mmu-miR-295 MI0000393(mmu-miR-295/295*)

On the sixth day after infection, the cells were harvested bytrypsinization and the whole amount of 5×10⁵ cells was spread over onmytomicin-C treated STO cells as feeder cells. Every other daythereafter, human ES cell medium containing bFGF (ReproCELL) wasreplaced. On the 24th, 32nd, and 40th day, the number of total coloniesand the number of colonies having morphology of human ES-like cells werecounted. As a control, DsRed was used in place of miRNA. The results areshown in FIG. 3. It was found that the number of colonies of inducedpluripotent stem cells increased twice or more, as compared to thecontrol, by introduction of three genes in the presence of hsa-miR-372,373, 302b, 302-367 cluster (including 302b, 302c, 302a, 302d, and 367),520c, mmu-miR-291a, 294, or 295.

Example 4 Preparation of Induced Pluripotent Stem Cells Through NuclearReprogramming of Adult Human Dermal Fibroblasts

3×10⁵ aHDF-Slc7a1 cells were plated on 60 mm gelatin coated dishes andinfected with retrovirus to express DsRed, 4 factors (OCT3/4, SOX2,c-MYC, and KLF4), or 3 factors (OCT3/4, SOX2, KLF4) in the presence ofvarious miRNAs independently. Six days after infection, 5×10⁵aHDF-Slc7a1 cells were reseeded on mytomicin-C treated STO cells. Fortydays after infection, the number of human ES-like colonies was counted.The same experiment was repeated three times.

FIG. 4A shows the results of three independent experiments. It was foundthat the number of colonies of induced pluripotent stem cells increased,as compared to the control, by introduction of three genes in thepresence of hsa-miR-372, 373/373*(hsa-miR-373), 371-373 cluster(including 371, 372, and 373), 302b/302b* (hsa-miR-302b), 302-367cluster (including 302b, 302c, 302a, 302d, and 367), 520c-5p/520c-3p(hsa-miR-520c), mmu-mir-290-5p/290-3p (mmu-mir-290),mmu-mir-291a-5p/291a-3p (mmu-mir-291a), 294/294* (mmu-mir-294), or295/295* (mmu-mir-295).

FIG. 4B shows the morphology of ES-like colonies of iPS cells by usingmicroscopy.

Example 5 Expression of ES Cell Markers in iPS Cells Produced by NuclearReprogramming of Mouse Tail Tip Fibroblasts (TTFs) with 4 Factors(OCT3/4, SOX2, c-MYC, and KLF4) and with 3 Factors (OCT3/4, SOX2, andKLF4)+mmu-miR-295/295*

5×10⁴ FbNg TTFs (TTFs derived from Fbx15-β geo/Nanog-IRES-Puro^(r)reporter mouse) cells were plated on gelatin coated 6-well plates andinfected with retrovirus to express 3 factors (Oct3/4, Sox2, Klf4) pluseither DsRed (Myc(−)3f+DsRed), mmu-miR-295/295*(Myc(−)3f+mmu-miR-295/295*), or c-Myc (4 factor). On Day 4 afterinfection, all the cells (Myc( )3f+DsRed; Myc(−)3f+mmu-miR-295/295*) or20 times diluted cells (4 factors) were reseeded on Puromycin andHygromycin-resistant-MSTO (PH-MSTO) cells. Puromycin selection wasstarted on Day 7, 14, 21, 28.

RT-PCR analysis using the Rever Tra Ace Kit (Takara) showed that the iPScells transfected with 4 factors (OCT3/4, SOX2, c-MYC, and KLF4), orwith 3 factors (OCT3/4, SOX2, and KLF4)+mmu-miR-295/295* expressed theES cell specific marker genes Oct3/4, Sox2, Nanog, and that the amountsof expression thereof were equivalent to those obtained with mouse EScells(ES) and mouse iPS cells (Fbx iPS) (FIG. 5).

Example 6 Preparation of Induced Pluripotent Stem Cells Through NuclearReprogramming of Mouse Embryonic Fibroblasts with 3 Factors (Oct3/4,Klf4, and c-Myc) with miRNAs

1×10⁵ Nanog MEFs (MEFs derived from Nanog-IRES-Puro^(r) reporter mouse)were plated on gelatin coated 6-well plates and infected with retrovirusto express 3 factors (Oct3/4, c-MycWT(wild type), and Klf4) withmmu-miR-290-295 cluster, 290-5p/290-3p (mmu-miR-290), 291a-5p/291a-3p(mmu-miR-291), 292-5p/292-3p (mmu-miR-292), 293/293* (mmu-miR-293),294/294* (mmu-miR-294) or 295/295* (mmu-miR-295) miRNAs (1:1). On day 4after infection, half of the cells were reseeded on Puromycin andHygromycin-resistant-MSTO (PH-MSTO) cells. Puromycin selection wasstarted from 14 days after infection.

FIG. 6A shows the number of Nanog GFP positive colonies. The results ofthree independent experiments are shown with different colors. “DsRed”indicates the combination of Oct3/4, Klf4, c-Myc and DsRed.

FIG. 6B shows the results of RT-PCR analysis. RT-PCR analysis using theRever Tra Ace Kit (Takara) showed that the iPS cells transfected with 4factors (OCT3/4, SOX2, c-MYC, and KLF4), or with 3 factors (OCT3/4,SOX2, and KLF4)+mmu-miR-290-295 cluster, 291a-5p/291a-3p, 294/294* and295/295* expressed the ES cell specific marker genes Oct3/4, Sox2,Nanog, and that the amounts of expression thereof were equivalent tothose obtained with mouse ES cells (ES) and mouse iPS cells (Fbx iPS).

Example 7 iPS Induction with Fb-Ng MEFs (MEFs Derived from Fbx15-βgeo/Nanog-IRES-Puro^(r) Reporter Mouse) Over-Expressing Oct3/4, c-Myc,and Klf4 (“Sox(−)”)+mmu-miR-295/295* or hsa-miR-302-367 Cluster miRNAs

1×10⁵ Fb-Ng MEFs (MEFs derived from Fbx15-β geo/Nanog-IRES-Puro^(r)reporter mouse) were plated on gelatin coated 6-well plates and infectedwith retrovirus to express 3 factors (Oct3/4, c-MycWT(wild type),Klf4)+miR-295/295*or hsa-miR-302-367 cluster. On day 4 after infection,cells were reseeded on Puromycin and Hygromycin resistant mytomycin-Ctreated STO cells (PH-MSTO) by in 6-well or 10 cm dishes. Puromycinselection was started from 7 days after infection.

FIG. 7A shows cell morphology of MEFs transduced with Oct3/4, c-Myc, andKlf4 (“Sox(−)”)+mmu-miR-295/295*. The colonies showed morphology similarto that of ES cells. FIG. 7B shows chimeras derived from iPS cellsinduced with Sox(−)3f+mmu-miR-295/295*.

FIG. 7C shows embryoid body (EB)-mediated in vitro differentiation byhuman iPS cells. Human iPS cells (61B1, 61N2) which were established bytransduction of 4 genes (OCT3/4, KLF4, SOX2, and c-MYC, i.e. “OSMK”) or3 genes (OCT3/4, KLF4, and c-MYC, i.e., “OMK(SOX(−)”)+hsa-miR-302-367cluster miRNA were plated on a low-binding dish, and embryoid bodieswere formed on 100 mm dishes in accordance with the method described inTakahashi et al., Cell 131:861-872, 2007. After culturing for 2 weeks,the cells were stained using an antibody against each of α-fetoprotein(R&D systems) which is a differentiation marker for endodermal cells,α-smooth muscle actin (DAKO) which is a differentiation marker formesodermal cells, and Glial Fibrillary Astrocytic Protein (GFAP) (DAKO)which is a differentiation marker for ectodermal cells. The expressionof each marker was confirmed by staining. Nuclei were stained withHoechst 33342 (Invitrogen).

Example 8 iPS Induction with 4 Factors (OCT3/4, SOX2, MYC, KLF4) or 3Factors (OCT3/4, MYC, KLF4, i.e., “SOX(−)3”) with and without VariousmiRNAs

3×10⁵ cells of aHDF-Slc7a1 cells were plated on 60 mm gelatin coateddishes and infected with retrovirus to express 4 factors: OCT3/4, SOX2,c-MYC, KLF4 (OSMK) or 3 factors: SOX(−)3factors (OMK) in the presence ofmiRNAs as indicated (OMK:mock or miRNAs=2.5:1.5), or with 2 factors:OCT3/4+KLF4 (OK) in the presence of miRNAs as indicated. Cells wereinfected with DsRed as control. Six days after infection, 5×10⁵aHDF-Slc7a1 cells were reseeded on mytomicin-C treated STO cells(MSTOcells). On Day 40 after infection, the number of ES-like colonieswas counted.

TABLE 3 shows the number of human ES(hES)-like colonies in aHDF-Slc7a1cells transduced with OSMK, OMK with or without miRNAs, and with OK withor without miRNAs. The hES-like colonies showed in cells transduced withOSMK, OMK+miRNAs (hsa-miR-371-373 cluster, hsa-miR-302-367 cluster, orhsa-miR-371-373 cluster+302-367 cluster) were detected by sixindependent experiments (Exp. 54, 61, 63, 114, 130, and 133).

TABLE 3 Number of hES-like colonies Exp. 54 Exp. 61 Exp. 63 Exp. 114Exp. 130 Exp. 133 A control DsRed 0 0 0 0 0 0 B OSMK Y4f (O:S:M:K =1:1:1:1) 5 41 54 37 39 100 G Y4f (OMK:S = 2.5:1.5) 13 7 4 22 H OMK +mock Sox(−)Y3f + mock 0 0 0 0 0 0 I or miRNA Sox(−)Y3f + h-miR-371-373cluster 0 0 1 0 0 0 J Sox(−)Y3f + h-miR-302-367 cluster 0 2 3 0 8 5 KSox(−)Y3f + h-mir-371-373 cluster + 302-367 0 0 6 cluster M OK + mockOK + mock 0 0 0 N or miRNA OK + h-miR-371-373 cluster 0 0 0 O OK +h-miR-302-367 cluster 0 0 4 P OK + h-miR-371-373 cluster + 302-367cluster 1 0 0

FIG. 8 shows cell morphology of iPS cells induced with OSMK (61B1); OMK(SOX(−))+hsa-miR-302-367 cluster miRNA (61N2); and OK+hsa-miR-302-367cluster miRNA (133O1).

INDUSTRIAL APPLICABILITY

The present invention provides an efficient method for preparing inducedpluripotent stem cells. The method of the present invention has highernuclear reprogramming efficiency as compared to conventional methods.For example, safe induced pluripotent stem cells can be efficientlyproduced without using c-Myc or gene products thereof. Accordingly, themethod of the present invention enables efficient production of highlysafe induced pluripotent stem cells from a patient's own somatic cells.Cells differentiated from such pluripotent stem cells (for example,myocardial cells, insulin-producing cells, or nerve cells) can be safelyutilized in stem cell transplantation therapies for treatment of variousdiseases, such as heart failure, insulin dependent diabetes mellitus,Parkinson's diseases, and spinal cord injury.

Although the present invention has been described in considerable detailwith regard to certain versions thereof, other versions are possible,and alterations, permutations, and equivalents of the versions shownwill become apparent to those skilled in the art upon a reading of thespecification and study of the drawings. Also, the various features ofthe versions herein can be combined in various ways to provideadditional versions of the present invention. Furthermore, certainterminology has been used for the purposes of descriptive clarity, andnot to limit the present invention. Therefore, any appended claimsshould not be limited to the description of the preferred versionscontained herein and should include all such alterations, permutations,and equivalents as fall within the true spirit and scope of the presentinvention.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the methods of the presentinvention can be carried out with a wide and equivalent range ofconditions, formulations, and other parameters without departing fromthe scope of the invention or any embodiments thereof.

All the disclosures of the above publication are incorporated herein byreference.

The attached Sequence Listing includes SEQ ID NOs: 13 and 14, as well asthose sequences disclosed in PCT/JP2008/59586, which is incorporated byreference herein in its entirety.

1. A method of preparing induced pluripotent stem cells, comprisingnuclear reprogramming at least one somatic cell with nuclearreprogramming factor and at least one miRNA, wherein the at least onemiRNA increases efficiency of the nuclear reprogramming of the at leastone somatic cell compared to nuclear reprogramming of the at least onesomatic cell with the nuclear reprogramming factor in the absence of theat least one miRNA.
 2. The method according to claim 1, wherein the atleast one miRNA is expressed in embryonic stem cells at a higher levelthan in somatic cells.
 3. The method according to claim 1, wherein agene encoding the nuclear reprogramming factor and/or the at least onemiRNA is introduced into the at least one somatic cell.
 4. The methodaccording to claim 3, wherein a vector comprising the gene and/or avector encoding the at least one miRNA is introduced into the at leastone somatic cell.
 5. The method according to claim 4, wherein the vectorcomprising the gene or encoding the at least one miRNA is a retroviralvector.
 6. The method according to claim 3, wherein the gene is selectedfrom an Oct family gene, a Klf family gene, and a Sox family gene. 7.The method according to claim 3, wherein the gene is selected fromOct3/4, Klf4, and Sox2.
 8. The method according to claim 3, wherein thenuclear reprogramming factor comprises Oct3/4, Klf4, and Sox2.
 9. Themethod according to claim 3, wherein the at least one miRNA isintroduced into the at least one somatic cell as primary miRNA.
 10. Themethod according to claim 3, wherein the at least one miRNA isintroduced into the at least one somatic cell as pre-miRNA.
 11. Themethod according to claim 1, wherein the at least one miRNA comprises atleast one miRNA represented by SEQ ID NOs: 1 to
 14. 12. The methodaccording to claim 1, wherein the at least one miRNA comprises at leastone miRNA contained in miRNA cluster hsa-miR-302-367 cluster,hsa-miR-371-373 cluster and hsa-miR-520c.
 13. The method according toclaim 1, wherein the at least one miRNA regulates DNA methylation. 14.The method according to claim 11, wherein the at least one miRNAregulates de novo DNA methylation.
 15. The method according to claim 11,wherein the at least one miRNA down-regulates DNA methylation.
 16. Themethod according to claim 1, wherein the at least one miRNA comprises atleast 10 contiguous nucleotides of at least one miRNA represented by SEQID NOs: 1 to
 14. 17. The method according to claim 1, wherein the atleast one miRNA comprises at least 30 contiguous nucleotides of at leastone miRNA represented by SEQ ID NOs: 1 to
 14. 18. The method accordingto claim 1, wherein the at least one miRNA comprises at least 60contiguous nucleotides of at least one miRNA represented by SEQ ID NOs:1 to
 14. 19. The method according to claim 1, wherein the nuclearreprogramming factor does not include c-Myc and/or Sox2.
 20. The methodaccording to claim 1, wherein the at least one miRNA compriseshsa-miR-302-367 cluster, hsa-miR-371-373 cluster and hsa-miR-520C miRNA.21. The method according to claim 20, wherein the nuclear reprogrammingfactor comprises an Oct family gene member, a Sox family gene member,and a Klf family gene member.
 22. The method according to claim 1,wherein the at least one miRNA comprises mmu-miR-295/295* and 294/294*.23. The method according to claim 1, wherein the at least one miRNAcomprises hsa-miR-302-367 cluster and hsa-miR-371-373 cluster miRNA. 24.The method according to claim 1, wherein the nuclear reprogrammingfactor comprises a Klf family gene, and an Oct family gene.
 25. Themethod according to claim 24, wherein the nuclear reprogramming factorfurther comprises a Myc family gene.
 26. The method according to claim24, wherein the nuclear reprogramming factor further comprises a Soxfamily gene.
 27. The method according to claim 25, wherein the nuclearreprogramming factor further comprises a Sox family gene.
 28. The methodaccording to claim 24, wherein the nuclear reprogramming factorcomprises KLF4 and OCT3/4.
 29. A method of increasing the efficiency ofnuclear reprogramming comprising: adding a nuclear reprogramming factorand at least one miRNA to at least one somatic cell so that the numberof induced pluripotent stem cells produced is greater than in theabsence of the added miRNA.
 30. An induced pluripotent stem cell inducedby reprogramming a somatic cell, wherein the reprogramming is performedby adding at least one miRNA and in the absence of eggs, embryos, orembryonic stem (ES) cells.
 31. The induced pluripotent stem cellaccording to claim 30, wherein the induced pluripotent stem cell is ahuman cell.
 32. An induced pluripotent stem cell obtained by the methodof claim
 1. 33. An induced pluripotent stem cell obtained by the methodof claim
 29. 34. A somatic cell derived by inducing differentiation ofthe pluripotent stem cell according to claim
 32. 35. A somatic cellderived by inducing differentiation of the pluripotent stem cell ofclaim
 33. 36. The method according to claim 25, wherein the nuclearreprogramming factor excludes a Sox family gene.
 37. The methodaccording to claim 26, wherein the nuclear reprogramming factor excludesa Myc family gene.
 38. The method according to claim 1, where the atleast one somatic cell comprises a plurality of somatic cells.